scholarly journals Restoration of superoxide generation to a chronic granulomatous disease- derived B-cell line by retrovirus mediated gene transfer

Blood ◽  
1992 ◽  
Vol 80 (5) ◽  
pp. 1125-1129 ◽  
Author(s):  
A Thrasher ◽  
M Chetty ◽  
C Casimir ◽  
AW Segal
Keyword(s):  
Nadph Oxidase ◽  
B Cell ◽  
Chronic Granulomatous Disease ◽  
Cell Line ◽  
B Lymphocyte ◽  
Single Gene ◽  
Granulomatous Disease ◽  
Inherited Disorders ◽  
Barr Virus ◽  
Epstein Barr

Failure of a superoxide generating system, the NADPH oxidase, present in neutrophils and other phagocytes gives rise to chronic granulomatous disease (CGD), a group of single-gene inherited disorders all characterized by an extreme susceptibility to pyogenic infection, with potentially fatal consequences. About 30% of CGD cases are caused by an autosomally inherited deficiency of a 47-Kd cytoplasmic component of the oxidase (p47-phox). Epstein-Barr virus (EBV) immortalized B- lymphocyte lines established from these CGD patients also express this NADPH oxidase defect and consequently are rendered incapable of generating superoxide on stimulation. We have used a p47-phox-deficient EBV-transformed B-cell line as a recipient for retroviral transfer of a functional p47-phox cDNA. The presence and activity of the retrovirally encoded p47-phox in the transduced cells is demonstrated and we show that this restores their capacity to generate superoxide.

scholarly journals Restoration of superoxide generation to a chronic granulomatous disease- derived B-cell line by retrovirus mediated gene transfer

Blood ◽  
1992 ◽  
Vol 80 (5) ◽  
pp. 1125-1129 ◽  
Author(s):  
A Thrasher ◽  
M Chetty ◽  
C Casimir ◽  
AW Segal
Keyword(s):  
Nadph Oxidase ◽  
B Cell ◽  
Chronic Granulomatous Disease ◽  
Cell Line ◽  
B Lymphocyte ◽  
Single Gene ◽  
Granulomatous Disease ◽  
Inherited Disorders ◽  
Barr Virus ◽  
Epstein Barr

Abstract Failure of a superoxide generating system, the NADPH oxidase, present in neutrophils and other phagocytes gives rise to chronic granulomatous disease (CGD), a group of single-gene inherited disorders all characterized by an extreme susceptibility to pyogenic infection, with potentially fatal consequences. About 30% of CGD cases are caused by an autosomally inherited deficiency of a 47-Kd cytoplasmic component of the oxidase (p47-phox). Epstein-Barr virus (EBV) immortalized B- lymphocyte lines established from these CGD patients also express this NADPH oxidase defect and consequently are rendered incapable of generating superoxide on stimulation. We have used a p47-phox-deficient EBV-transformed B-cell line as a recipient for retroviral transfer of a functional p47-phox cDNA. The presence and activity of the retrovirally encoded p47-phox in the transduced cells is demonstrated and we show that this restores their capacity to generate superoxide.

scholarly journals X-linked chronic granulomatous disease: correction of NADPH oxidase defect by retrovirus-mediated expression of gp91-phox

Blood ◽  
1993 ◽  
Vol 82 (7) ◽  
pp. 2196-2202 ◽  
Author(s):  
CD Porter ◽  
MH Parkar ◽  
RJ Levinsky ◽  
MK Collins ◽  
C Kinnon
Keyword(s):  
Gene Therapy ◽  
Nadph Oxidase ◽  
B Cell ◽  
Chronic Granulomatous Disease ◽  
Cell Lines ◽  
Large Subunit ◽  
Granulomatous Disease ◽  
Barr Virus ◽  
Inherited Immunodeficiency ◽  
Epstein Barr

Chronic granulomatous disease (CGD) is an inherited immunodeficiency resulting from the inability of an individual's phagocytes to produce superoxide anions because of defective NADPH oxidase. The disease may be treated by bone marrow transplantation and as such is a candidate for somatic gene therapy. Two thirds of patients have defects in an X- linked gene (X-CGD) encoding gp91-phox, the large subunit of the membrane cytochrome b-245 component of NADPH oxidase. Epstein-Barr virus-transformed B-cell lines from patients with CGD provide a model system for the disease. We have used retrovirus-mediated expression of gp91-phox to reconstitute functionally NADPH oxidase activity in B-cell lines from three unrelated patients with X-CGD. The protein is glycosylated and membrane associated, and the reconstituted oxidase is appropriately activated via protein kinase C. The kinetics of superoxide production by such reconstituted cells is similar to that of normal B-cell lines. These data show the potential of gene therapy for this disease.

scholarly journals X-linked chronic granulomatous disease: correction of NADPH oxidase defect by retrovirus-mediated expression of gp91-phox

Blood ◽  
1993 ◽  
Vol 82 (7) ◽  
pp. 2196-2202 ◽  
Author(s):  
CD Porter ◽  
MH Parkar ◽  
RJ Levinsky ◽  
MK Collins ◽  
C Kinnon
Keyword(s):  
Gene Therapy ◽  
Nadph Oxidase ◽  
B Cell ◽  
Chronic Granulomatous Disease ◽  
Cell Lines ◽  
Large Subunit ◽  
Granulomatous Disease ◽  
Barr Virus ◽  
Inherited Immunodeficiency ◽  
Epstein Barr

Abstract Chronic granulomatous disease (CGD) is an inherited immunodeficiency resulting from the inability of an individual's phagocytes to produce superoxide anions because of defective NADPH oxidase. The disease may be treated by bone marrow transplantation and as such is a candidate for somatic gene therapy. Two thirds of patients have defects in an X- linked gene (X-CGD) encoding gp91-phox, the large subunit of the membrane cytochrome b-245 component of NADPH oxidase. Epstein-Barr virus-transformed B-cell lines from patients with CGD provide a model system for the disease. We have used retrovirus-mediated expression of gp91-phox to reconstitute functionally NADPH oxidase activity in B-cell lines from three unrelated patients with X-CGD. The protein is glycosylated and membrane associated, and the reconstituted oxidase is appropriately activated via protein kinase C. The kinetics of superoxide production by such reconstituted cells is similar to that of normal B-cell lines. These data show the potential of gene therapy for this disease.

scholarly journals Detection of gp91-phox precursor protein in B-cell lines from patients with X-linked chronic granulomatous disease as an indicator for mutations impairing cytochrome b558 biosynthesis

1996 ◽  
Vol 315 (2) ◽  
pp. 571-575 ◽  
Author(s):  
Colin D. PORTER ◽  
KURIBAYASHI KURIBAYASHI ◽  
Mohamed H. PARKAR ◽  
Dirk ROOS ◽  
Christine KINNON
Keyword(s):  
Nadph Oxidase ◽  
B Cell ◽  
Chronic Granulomatous Disease ◽  
Cell Lines ◽  
Binding Domain ◽  
Granulomatous Disease ◽  
Cytochrome B558 ◽  
Stable Association ◽  
P22 Phox ◽  
Fad Binding

NADPH oxidase cytochrome b558 consists of two subunits, gp91-phox and p22-phox, defects of which result in chronic granulomatous disease (CGD). The nature of the interaction between these subunits has yet to be determined. Absence of p22-phox in autosomal CGD patient-derived B-cell lines results in detectable levels of an incompletely glycosylated gp91-phox precursor. We have detected this same precursor species in four cell lines from patients with the X-linked form of the disease due to mutations in gp91-phox. Such mutations should delineate regions of gp91-phox important for its biosynthesis, including stable association with p22-phox. One mutation mapped to the putative FAD-binding domain, one mapped to a potential haem-binding domain, and two involved the region encoded by exon 3.

scholarly journals Transcription of HLA class II genes in the absence of B-cell-specific octamer-binding factor.

1988 ◽  
Vol 8 (9) ◽  
pp. 3734-3739 ◽  
Author(s):  
E Stimac ◽  
S Lyons ◽  
D Pious
Keyword(s):  
T Cell ◽  
B Cell ◽  
Cell Lines ◽  
B Lymphocyte ◽  
Class Ii ◽  
Cell Mutant ◽  
Barr Virus ◽  
Binding Factor ◽  
Epstein Barr ◽  
T Cell Lines

HLA-DR and other human class II histocompatibility genes are expressed by Epstein-Barr virus-transformed B-lymphocyte cell lines but not by most T-cell leukemia lines. We determined by transcriptional run-on experiments that regulation of class II expression in these cells is at the level of gene transcription; nuclei isolated from B-cell lines actively transcribe class II mRNA, whereas nuclei from non-class II-expressing T-cell lines and from the class II transactive factor-deficient B-cell mutant 6.1.6 do not. In searching for DNA-binding proteins which might regulate transcription, we found both a ubiquitous (B1) and a B-cell-specific (B2) factor which bind to the octamer sequence ATTTGCAT 52 base pairs 5' of the cap site in the DR alpha gene. We examined the relationship of these factors to DR alpha transcription. HUT-78, a T-cell line which expresses class II mRNA constitutively, contains only the ubiquitous B1 octamer-binding factor also found in non-class II-expressing T-cell leukemias. Human fibroblast, HeLa, and melanoma cell lines similarly contain only the ubiquitous factor, even when these cells are induced to express class II mRNA by treatment with gamma interferon. Both B1 and B2 binding factors are present in the B-cell mutant 6.1.6, which nevertheless fails to transcribe class II mRNA. Although we have not ruled out the requirement of B-cell-specific octamer-binding factor B2 for class II expression in B cells, it is clear that in other cells substantial DR alpha transcription occurs in the absence of this factor.

Transcription of HLA class II genes in the absence of B-cell-specific octamer-binding factor

1988 ◽  
Vol 8 (9) ◽  
pp. 3734-3739
Author(s):  
E Stimac ◽  
S Lyons ◽  
D Pious
Keyword(s):  
T Cell ◽  
B Cell ◽  
Cell Lines ◽  
B Lymphocyte ◽  
Class Ii ◽  
Cell Mutant ◽  
Barr Virus ◽  
Binding Factor ◽  
Epstein Barr ◽  
T Cell Lines

HLA-DR and other human class II histocompatibility genes are expressed by Epstein-Barr virus-transformed B-lymphocyte cell lines but not by most T-cell leukemia lines. We determined by transcriptional run-on experiments that regulation of class II expression in these cells is at the level of gene transcription; nuclei isolated from B-cell lines actively transcribe class II mRNA, whereas nuclei from non-class II-expressing T-cell lines and from the class II transactive factor-deficient B-cell mutant 6.1.6 do not. In searching for DNA-binding proteins which might regulate transcription, we found both a ubiquitous (B1) and a B-cell-specific (B2) factor which bind to the octamer sequence ATTTGCAT 52 base pairs 5' of the cap site in the DR alpha gene. We examined the relationship of these factors to DR alpha transcription. HUT-78, a T-cell line which expresses class II mRNA constitutively, contains only the ubiquitous B1 octamer-binding factor also found in non-class II-expressing T-cell leukemias. Human fibroblast, HeLa, and melanoma cell lines similarly contain only the ubiquitous factor, even when these cells are induced to express class II mRNA by treatment with gamma interferon. Both B1 and B2 binding factors are present in the B-cell mutant 6.1.6, which nevertheless fails to transcribe class II mRNA. Although we have not ruled out the requirement of B-cell-specific octamer-binding factor B2 for class II expression in B cells, it is clear that in other cells substantial DR alpha transcription occurs in the absence of this factor.

scholarly journals Interferon-gamma improves splicing efficiency of CYBB gene transcripts in an interferon-responsive variant of chronic granulomatous disease due to a splice site consensus region mutation

Blood ◽  
2000 ◽  
Vol 95 (11) ◽  
pp. 3548-3554 ◽  
Author(s):  
Antonio Condino-Neto ◽  
Peter E. Newburger
Keyword(s):  
B Cell ◽  
Chronic Granulomatous Disease ◽  
Cell Line ◽  
Interferon Gamma ◽  
Granulomatous Disease ◽  
First Intron ◽  
Cybb Gene ◽  
Nuclear Rna ◽  
Ifn Γ

Abstract X-linked chronic granulomatous disease (CGD) derives from defects in the CYBB gene, which encodes the gp91-phox component of NADPH oxidase. We studied the molecular basis of the disease in a kindred with variant CGD, due to a single base substitution at the sixth position of CYBB first intron. The patients' phagocytes have been shown previously to greatly increase superoxide release in response to interferon-gamma (IFN-γ) in vitro and in vivo. We examined CYBB gene expression in an Epstein-Barr virus (EBV)-transformed B-cell line from 1 patient in this kindred. These cells showed markedly decreased levels of CYBB transcripts in total RNA (5% of normal) and nuclear RNA (1.4% of normal), despite equal CYBB transcription rates in the CGD and control cells. Incubation with IFN-γ produced a 3-fold increase in CYBBtotal messenger RNA (mRNA) levels in the patient's cells, and decreased nuclear transcripts to undetectable levels. Reverse transcriptase–polymerase chain reaction analysis of RNA splicing revealed a preponderance of unspliced CYBB transcripts in the patient's nuclear RNA. In vitro incubation with IFN-γ increased by 40% the ratio of spliced relative to unspliced CYBB mRNA in nuclei from the CGD B-cell line. Total RNA harvested from the same patient's monocytes, on and off therapy with IFN-γ, showed a similar improvement in splicing. We conclude that IFN-γ partially corrects a nuclear processing defect due to the intronic mutation in theCYBB gene in this kindred, most likely by augmentation of nuclear export of normal transcripts, and improvement in the fidelity of splicing at the first intron.

scholarly journals Signatures of B-cell receptor diversity in B lymphocytes following Epstein-Barr virus transformation

2019 ◽  
Vol 51 (6) ◽  
pp. 197-207
Author(s):  
Meimei Lai ◽  
Qiongdan Wang ◽  
Yutian Lu ◽  
Xi Xu ◽  
Ying Xia ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Epstein Barr Virus ◽  
High Throughput Sequencing ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Human Virus ◽  
Barr Virus ◽  
Ebv Infection ◽  
Epstein Barr

Epstein-Barr virus (EBV) is a widespread human virus that establishes latent infection, potentially leading to tumors, hematological disorders, and other severe diseases. EBV infections are associated with diverse symptoms and affect various organs; therefore, early diagnosis and treatment are crucial. B cell receptor (BCR) repertoires of B cell surface immunoglobulins have been widely studied for their association with various infectious diseases. However, the specific genetic changes that modulate the BCR repertoires after an EBV infection are still poorly understood. In this study, we employed high-throughput sequencing (HTS) to investigate the diversity of BCR repertoires in an EBV-transformed lymphoblastic cell line (LCL). Compared with the noninfected control B cell line, the LCL exhibited a decrease in overall BCR diversity but displayed an increase in the expansion of some dominant rearrangements such as IGHV4-31/IGHJ4, IGHV4-59/IGHJ4, IGHV5-51/IGHJ3, and IGHV3-74/IGHJ3. A higher frequency of occurrence of these rearrangement types was confirmed in patients with EBV infection. Interestingly, the IGHV3-74 rearrangement was only detected in EBV-infected children, suggesting that our experimental observations were not coincidental. In addition, we identified a highly dominant consensus motif, CAR(xRx)YGSG(xYx)FD, in complementarity-determining region 3 (CDR3) sequences of the heavy chain in the LCL. Our findings demonstrated the utility of HTS technology for studying the variations in signature motifs of the BCR repertoires after EBV infection. We propose that the analysis of BCR repertoire sequences represents a promising method for diagnosing early EBV infections and developing novel antibody- and vaccine-based therapies against such infections.

scholarly journals CD34+ peripheral blood progenitors as a target for genetic correction of the two flavocytochrome b558 defective forms of chronic granulomatous disease

Blood ◽  
1994 ◽  
Vol 84 (1) ◽  
pp. 53-58 ◽  
Author(s):  
F Li ◽  
GF Linton ◽  
S Sekhsaria ◽  
N Whiting-Theobald ◽  
JP Katkin ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Chronic Granulomatous Disease ◽  
Peripheral Blood ◽  
B Lymphocyte ◽  
Leukemia Virus ◽  
Single Gene ◽  
Fold Increase ◽  
Granulomatous Disease ◽  
Flavocytochrome B558 ◽  
O2 Production

Abstract Chronic granulomatous disease (CGD) can result from any of four single gene defects involving components of the superoxide (O2-.)-generating phagocyte NADPH oxidase (phox). The phox transmembrane flavocytochrome b558 is composed of two peptides, gp91phox and p22phox. Mutations of gp91phox cause X-linked CGD, whereas mutations of p22phox cause one of the three autosomal recessive forms of CGD. We used the Maloney leukemia virus-based MFG retrovirus vector to produce replication defective retroviruses encoding gp91phox or p22phox. To maximize viral titer MFG retroviruses do not contain internal promoter or resistance elements. Epstein-Barr virus transformed B-lymphocyte cell lines (EBV- B) derived from normal individuals contain phox components and produce O2-., whereas those derived from CGD patients show the CGD defect. Transduction of gp91phox or p22phox-deficient CGD EBV-B lines resulted in correction of O2-. production from a barely detectable baseline to an average 7.2% and 13.8% of normal control, respectively, without any selective regimen to enrich for transduced cells. CD34+ hematopoietic progenitor cells, the therapeutic target for gene therapy of CGD, were isolated from peripheral blood of CGD patients, transduced with MFG- phox retroviruses, and differentiated in culture to mature phagocytes. Transduction of progenitors corrected the gp91phox (seven patients) and p22phox (two patients) CGD phagocyte oxidase defect to 2.5% and 4.9% of normal O2-. production, respectively, representing an 87-fold and 161- fold increase. These studies show correction of flavocytochrome b558- deficient CGD in primary hematopoietic progenitors, providing a basis for development of gene therapy for the X-linked gp91phox and autosomal p22phox-deficient forms of CGD.

Sign in / Sign up

Export Citation Format

Share Document